Correlation between histopathological and FT-Raman spectroscopy analysis of the liver of Swiss mice infected with Paracoccidioides brasiliensis.

Paracoccidioidomycosis is the most important systemic mycosis in Latin America. The main entrance of the fungus is the airway. It primarily occurs in the lung, but in its disseminated form may affect any organ. The liver is one of the organs afflicted by this disease and its homeostasis may be impaired. The aim of the present study was to evaluate the evolution of paracoccidioidomycosis in the liver of Swiss mice and correlate morphological factors with the expression of gp43 and with physicochemical analysis via FT-Raman of the infected organ. According to colony forming unit (CFU) and granuloma counting, the first and second weeks were the periods when infection was most severe. Tissue response was characterized by the development of organized granulomas and widespread infection, with yeasts located within the macrophages and isolated hepatocytes. The gp43 molecule was distributed throughout the hepatic parenchyma, and immunostaining was constant in all observed periods. The main physicochemical changes of the infected liver were observed in the spectral ranges between 1700-1530 cm(-1) and 1370-1290 cm(-1), a peak shifting center attributed to phenylalanine and area variation of -CH2 and -CH3 compounds associated to collagen, respectively. Over time, there was a direct proportional relationship between the number of CFUs, the number of granulomas and the physicochemical changes in the liver of mice infected with Paracoccidioides brasiliensis. The expression of gp43 was similar in all observed periods

Photomicrograph of: (A and B) liver of mice control, non-infected and (C and D) after eight weeks of infection with <i>Paracoccidiodes brasiliensis</i>.

<p>Note an increase in the number of birefringent collagen fibers (C). Immunhistochemistry using anti- gp43 antibody showed diffuse cytoplasmic and nuclear staining in the liver parenchyma (D). Stain: Picrosirius technique (A and C); Avidin-biotin peroxidase immunohistochemical using anti-gp43 antibody (B and D).</p

Number of Colony Forming Units (CFUs)/g in the liver of mice infected with <i>Paracoccidioides brasiliensis</i>.

<p>Results represent mean ± EPM and were expressed as Log₁₀; n = 6. Different letters represent P<0.001. ANOVA, Tukey post-test.</p

Raman spectrum from liver of mice (A) control, non-infected and (B and C) after one, two, four and eight weeks of infection with <i>Paracoccidioides brasiliensis</i>.

<p>The insets show the spectral range between 1750-1520 cm⁻¹ (B) and 1380-1200 cm⁻¹ (C) where the spectral difference compared non-infected with infected tissues were observed.</p

Non-infected mice liver Raman bands and its assignments.

<p>v: stretching vibration; v_as: asymmetric stretching vibration; v_s: symmetric stretching vibration; δ_as: asymmetric bending vibration; δ_s: symmetric bending vibration.</p><p>Non-infected mice liver Raman bands and its assignments.</p

Comparison of area ratio (1340 cm⁻¹/1260 cm⁻¹) between mice non-infected liver tissue (t = 0) and after one, two, four and eight weeks of <i>Paracoccidioides brasiliensis</i> infection.

<p>Comparison of area ratio (1340 cm⁻¹/1260 cm⁻¹) between mice non-infected liver tissue (t = 0) and after one, two, four and eight weeks of <i>Paracoccidioides brasiliensis</i> infection.</p

Photomicrograph of: (A) liver of mice control, non-infected and (B - F) after one week of infection with <i>Paracoccidiodes brasiliensis</i>.

<p>In (B) yeasts were not observed in the stained granulomas with H&E, though sprouting and varied shapes were indentified with (C) Gomori-Grocott staining; in (D) and (E) note the presence of fungical fragments impregnated with silver within hepatocytes and macrophages (arrow); in (F) large number of mitosis (arrow) were observed in the hepatic parenquima. Stain: H & E (A and B); Gomori-Grocott counter-stained with green light (C and E) or H & E (D); Periodic acid-Schiff (PAS)(F).</p

Photomicrograph of: (A and B) liver of mice control, non-infected and (C - F) after four (G and H) and eight weeks of infection with <i>Paracoccidiodes brasiliensis</i>.

<p>Organized granulomas (C) with a fewest or without yeasts (C-F) were observed on the fourth week. On the eighth week yeasts were still observed. Stain: Periodic acid-Schiff (PAS) (A and C); Gomori-Grocott counter-stained with green light (B and E-F) or H&E (D and H).</p

(A) Shifting of 1615 cm⁻¹ peak position observed in non-infected liver (t = 0) toward low frequencies as a function of time infection.

<p>(B) Behavior of amino acids band area (1710-1510 cm⁻¹) as a function of time infection.</p